1. Field of the Invention
This invention relates to a method and apparatus, including component devices, for use in automating the detection of target nucleic acid sequences in biological-containing samples. The method involves a sequence of physical and chemical reactions, and more particularly to a system for the exposure of, amplification of, and labelled-probe coupling to, a specific, known nucleic acid sequence. The process of the invention consists of the following stages: 1) matrix dispensing, sample mixing and DNA immobilization; 2) preparing DNA; 3) amplifying DNA target sequences; 4) hybridizing a labeled probe to the target; and 5) scanning the matrices for signal produced by bound label.
The invention is especially suited to the automated detection of single, specific genetic sequences present at random in multiple samples containing biological material without labor-intensive DNA extraction and purification procedures being performed separately on each sample. The ability to detect single copies of a specific nucleic acid in biological or environmental samples makes this process revolutionary.
2. Description of the Related Art
There is no laboratory apparatus or equipment currently on the market that automates DNA preparation, modification and detection in one, unattended operation. The apparatus and devices described herein embody an automated process, including a fluid-delivery system and a thermal reaction chamber.
Devices for receiving biological specimens for diagnostic purposes are varied and adapted to the methods of detection. The devices may take the form of tubes for liquid specimens, flat surfaces such as glass slides suitable for microscopy, microtiter dishes, Petri dishes and cubes containing growth medium, or filters made of various materials to which cell and viral components will adhere.
These specimen samples are then treated in such a way as to indicate either the presence or absence, or quantity, of a specific biological entity. Test reagents may either be preapplied to the device or added in series after the specimen is present. Test results are read manually by a technical person or automatically with instrumentation specifically designed for that assay. In some instances the specimen is diluted with a diluent, or an aliquot of the specimen is removed from the original collecting device and transferred to another vessel at some point in the assay. In some cases physical and chemical means are used to amplify the signal of the assay for greater sensitivity. Some assays require extraction or separation to isolate a specific component from other parts.
In DNA-based diagnostics the sequence specificity of base-pairing or enzymatic or other types of cleavage is exploited. The linear sequence of nucleotides in double-stranded DNA molecules forms the basis of replication of the genetic code. Hybridization is the binding of two single-stranded DNA strands whose base-pairing sequences are complementary. Temperature and salt concentration affect the stringency of these base-pairing matches. A change from high stringency to low stringency can cause the same DNA probe to be either exquisitely specific to detect a particular target or less specific and detect a group of related targets.
In some instances the sizes of DNA fragments, produced by restriction endonuclease digestion or by amplification of a target sequences between primer pairs, are used to make a DNA-print for individual identification or aid in diagnosis of a genetic disease, cancer or infectious disease. For example, electrophoresis may be used to size-fractionate different-sized nucleic acids which have been specifically cleaved or whose native length puts them in a distinguishable size-length class.
In the electrophoresis method, a current is applied to DNA loaded at the cathodal end of a gel matrix, which causes the DNA to migrate towards the anodal end of the matrix. The electrophoretic mobility of DNA is dependent on fragment size and is fairly independent of base composition or sequence. Resolution of one size class from another is better than 0.5% of fragment size (Sealy P. G. and E. M. Southern. 1982. Gel electrophoresis of DNA, p. 39-76. In D. Rickwood and B. D. Hames (EDS.), Gel Electrophoresis of Nucleic Acids. IRL Press, London). This reference and all other publications or patents cited herein are hereby incorporated by reference.
Electrophoresis methods thus require a vessel to hold the matrix material and the biological specimens to be subjected to electrophoresis. Such vessels may mold the gel matrix during its formation and may hold it during processing.
Diffusion of reagents is faster where the ratio of the matrix surface area to matrix volume is greatest as in thin, flat matrices. Likewise, electrophoresis of macromolecules requires less voltage and is faster in ultra-thin matrices or tiny (glass) capillaries. In these aqueous matrices, the vessel is necessary to prevent evaporation and to add strength in handling. Existing vessels that enclose matrices impede rapid diffusion of reagents and molecular probes. Once the existing vessels are taken apart in processing, they cannot be put back together to continue automated processing.
Accordingly, the invention aims to provide a system for automated gene identification of multiple samples, which prepares nucleic acids in the samples for testing, sufficiently amplifies target nucleic acid sequences and accurately detects their presence or absence in the samples.
Another object of the invention is to provide a carrier to contain specimens and be used as the sole vessel for completion of all steps of an assay, including sample preparation, electrophoresis, amplification and hybridization.
Yet another object of the invention is to provide support of the matrix and specimen, molding the matrix and embedding the specimen in it for automated processing.
A further object of the invention is to provide such a system which is adaptable to dispensing different quantities of different reagents for saturating specimens quickly with a series of solutions automatically.
A further object of the invention is to provide such a system wherein airflow and heating regulate and monitor temperature and humidity in the matrices including drying them.
A further object of the invention is to provide a system which can accommodate partial capacity loads, i. e., fewer matrices per run, or that can accommodate more than one probe per run.
A further object of the invention is to provide an automatic process and apparatus allowing identification of nucleic acid sequences that have been embedded or fractionated in a matrix whether or not prior extraction or purification of DNA has been performed in the invention.
A further object of the invention is to carry the specimen in transport from the point of collection to the processing point.
A further object of the invention is to provide a convenient way to make the particles containing target nucleic acids of a specimen in a matrix available and sufficiently spread for signal detection in a two-dimensional array.
A further object of the invention is to concentrate specimen nucleic acids, or amplified products thereof, for detection of their presence.
A further object of the invention is provide a barrier to evaporation of solutions during processing.
A further object of the invention is a mechanism to change configuration of the carriers during processing of the specimen to adapt to processing conditions.
A further objective of the invention is to provide support for reading the test results.
A still further object of the invention is to permanently store the nucleic acids present in the specimen for possible retesting and serve as a permanent record of the test, if an archival record is desired.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.